This application addresses broad Challenge Area (11) Regenerative Medicine - 11-HL-101* To develop stem cell based therapies for cardiovascular, lung and blood diseases. There Will be Blood: Stem cell niche driven derivation of HSC from ES cells Transplantation of hematopoietic stem cells (HSCs) derived from high-quality sources of tissue-matched bone marrow, mobilized peripheral blood or umbilical cord blood represents the therapeutic option of choice for the treatment of hematological malignancies and an increasing array of non-malignant blood disorders. Suitable bone marrow is often in short supply, and cord blood, although bankable, contains much lower numbers of HSCs, which makes it less suitable for adult transplantation. Directed differentiation of human embryonic stem (hES) cells toward HSCs offers a potentially attractive alternative to these conventional sources. The recent reports of somatic cell reprogramming by means of induced pluripotency makes this approach an even more exciting prospect for future cell-based therapies. The use of HSC derived from human embryonic stem cells (hESCs) as a therapeutic alternative to the traditional sources of reconstituting cells listed above is limited by the low efficiency of derivation protocols that have been described to date. The ontogeny of the mammalian hematopoietic system is marked by a temporally regulated migration of primitive HSC from their site of emergence in the early embryo via a period of expansion in the fetal liver to the bone marrow, the site of hematopoiesis throughout adult life. Thus following their specification and emergence from primitive mesoderm, the subsequent proliferation and maintenance of HSC occurs in association with a series of discrete microenvironmental niches whose cellular composition and properties vary with the organ of residence. This proposal will test the hypothesis that efficient in vitro derivation of transplantable HSC with long term repopulating potential from pluripotent stem cell sources (ES and iPS) will require exposure of the differentiating pluripotent cells to elements of these distinct microenvironmental niches in a manner that recapitulates both the temporal and organ-specific ontogeny of the hematopoietic system. Specific aims of this proposal are: 1.) To use a novel WNT- dependent protocol in chemically defined medium for the mesoderm specification of hES cells. To achieve stringent exogenous control over mesoderm specification we will utilize a recently optimized system driven by Wnt. The power approach will be further enhanced by the use of (i) a human ES line engineered to express GFP driven by the hMIXL promoter to allow identification of Flk-1+ hematopoietic mesoderm;(ii) additional cell surface markers of HSC identified in the PI's lab (including ACE/CD143 and PODXL). 2.) To culture the mesoderm-fated progeny of hES cells in the presence of stromal cell populations which temporally recapitulate stem cell niches present during hematopoietic ontogeny ES-derived populations of interest (dependent on Aim 1) will be co-cultured in sequence under various conditions with conditionally immortalised stromal cell lines (derived from the Immortomouse) previously generated in the PI's lab from the AGM, fetal liver and adult bone marrow (osteoblast and vascular niches). Primitive hematopoietic cells generated under each condition will be assayed for their immunophenotypic properties and content of clonogenic hematopoietic progenitor cells. Cells generated under conditions that best promote the emergence of primitive hematopoietic cells will be assayed for their content of HSC by intrafemoral injection into immunodeficient NOD/LtSz-Scid IL2R null mice. HSC content will be quantitated by limitdilution and serial transplants. The strength of this proposal lies in the consortium of investigators who collectively bring considerable expertise in the identification of biomarkers of human HSC and the stromal cell regulation of hematopoiesis (Simmons;PI), the mesodermal specification of hES cells (Nakayama) and in the derivation and molecular analysis of stromal cell lines (Brouard). PUBLIC HEALTH RELEVANCE: Transplantation of hematopoietic stem cells (HSCs) derived from high-quality sources of tissue-matched bone marrow, mobilized peripheral blood or umbilical cord blood currently represents the therapeutic option of choice for the treatment of hematological malignancies and an increasing array of non-malignant blood disorders. The limited supply of suitably matched HSC from these sources is a significant limitation to the safety and broader utility of haematological transplantation, prompting the search for alternative sources of HSC. This proposal seeks to develop markedly improved strategies to derive HSC from pluripotent stem cells as a means to overcome this limitation and to further the development of novel cellular therapies for treating blood diseases.